Compositions and methods useful for targeting blood-brain barrier

Abstract

To provide compositions and methods to improve delivery of gene therapy vectors and therapeutics across the BBB to provide more effective treatment for a variety of CNS conditions.SOLUTION: Compositions and methods for delivering effector entities to the CNS of a subject are provided. Engineered AAV capsids that bind GPI-anchored proteins on the BBB are provided as well as methods for their use, including delivery of gene therapy and effector entities. Also provided are methods for reducing the infectivity of the CNS by an AAV.SELECTED DRAWING: None

Description

[Background technology]

[0001] Gene therapy has been successfully advanced to the clinic for the treatment of several rare monogenic diseases. Vector platforms based on natural isolates of adeno-associated virus (AAV) have been essential for this success. A natural variant of AAV derived from human myocardium, called AAV9 (Gao et al. J Virol 78, 6381-6388, 2004; Bell et al. J Clin Invest 121, 2427-2435, 2011), shows excellent distribution after intravenous delivery (Zincarelli et al. Mol Ther 16, 1073-1080, 2008; Duque et al. Mol Ther 17, 1187-1196, 2009; Bevan et al. Mol Ther 19, 1971-1980, 2011). AAV9-based vectors have also been used to target motor neurons in patients with spinal muscular atrophy, resulting in improved motor function and prolonged survival (Mendell et al. Engl J Med 377, 1713-1722, 2017). In the treatment of children with rare genetic myopathies, similarly impressive results have been obtained by intravenously delivering vectors of the AAV8 serotype isolated from macaques (Gao et al. Proc Natl Acad Sci USA 99, 11854-11859, 2002) (NCT03199469). Despite improvements in AAV vectors with the isolation of variants such as AAV8 and AAV9, most candidate diseases have not been successful in in vivo gene therapy due to limited delivery to cells of target tissues.

[0002] Efficient delivery of gene therapy vectors across the blood-brain barrier (BBB) ​​is essential for the therapy of neurological diseases. However, adequate delivery levels have yet to be achieved. To improve AAV vector performance, various approaches have been pursued, including engineering capsid variants with improved efficiency. One engineering strategy is to generate diversity of capsid structures through population mutagenesis and then select favorable candidates by screening populations of cells or animals. The most widely known engineered AAV variant, called AAV-PHP.B, was shown to have excellent neurotropic properties (Deverman et al. Nat Biotechnol 34, 204-209, 2016). AAV-PHP.B was identified by generating a library of variants by inserting a population of seven amino acid domains into the hypervariable region VIII of AAV9. Variants that target the CNS were identified by intravenous injection into astrocyte-specific CRE recombinase mice on a C57BL / 6J background. From this selection, AAV-PHP.B was found, whose capsid contains the TLAVPFK domain and shows a 50-fold improvement in CNS transduction after intravenous delivery to C57BL / 6J mice (Deverman et al. Nat Biotechnol 34, 204-209, 2016). This transduction level has the potential to broaden the utility of AAV gene therapy for human neurological disorders.

[0003] There remains a need to improve the delivery of gene therapy vectors and therapeutic agents across the BBB to provide more effective treatments for a variety of CNS conditions. Summary of the Invention

[0004] The embodiments described herein relate to compositions and methods for improving the delivery of gene therapy vectors and effector entities to the CNS of a subject in need thereof. Methods for modifying AAV infectivity of the CNS are also provided.

[0005] In one aspect, provided herein is a composition comprising a recombinant AAV having a capsid, the capsid comprising a binding partner for a GPI-anchored blood-brain barrier (BBB) ​​ligand, and In certain embodiments, the ligand is conjugated to an effector entity. In certain embodiments, the ligand is Ly6E. In another aspect, the ligand is selected from GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

[0006] In one aspect, the AAV capsid is an empty capsid. In another embodiment, the capsid comprises an AAV vector genome encoding a heterologous gene.

[0007] In yet another embodiment, the effector entity is a peptide, a nucleic acid, a siRNA, an antibody, an antibody fragment, a small molecule, a lipid nanoparticle, or a cytotoxic agent. In one embodiment, the AAV capsid and the effector entity are conjugated via a linker.

[0008] In another embodiment, provided herein is a method for treating a neurological disease or disorder in a subject in need of such treatment, comprising contacting the BBB of the subject with an AAV having a capsid that includes a binding partner for a GPI-anchored BBB ligand conjugated to an effector entity, wherein the capsid binds to the GPI-anchored BBB ligand and mediates transport of the effector entity across the BBB. In one embodiment, the ligand is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B. In another aspect, the neurological disease or disorder is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, lysosomal storage disease, and traumatic brain injury.In certain embodiments, the effector entity is a peptide, a nucleic acid, an siRNA, an antibody, an antibody fragment, a small molecule, or a cytotoxic agent.In yet another embodiment, the AAV capsid is conjugated to the effector entity via a linker.

[0009] In yet another embodiment, a combination therapy is provided herein, the combination therapy reduces or inhibits central nervous system uptake of a gene therapy vector having a binding partner for a GPI-anchored BBB ligand and an AAV capsid, comprising co-administering an antibody or antibody fragment that binds to the BBB ligand with the gene therapy vector. In one aspect, the antibody binds to a ligand selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

[0010] In another aspect, provided herein is a method of engineering an AAV capsid to target the CNS, comprising: a) identifying an amino acid sequence encoding a peptide fragment that specifically binds to a GPI-anchored BBB ligand; and b) modifying an AAV HVRVIII site to express the amino acid sequence, wherein the engineered capsid binds to the GPI-anchored BBB ligand. In certain embodiments, the ligand is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1 and SEC24B. In another embodiment, the modified AAV is AAV1, AAV3B or AAV9.

[0011] In yet another aspect, provided herein is an engineered AAV capsid obtained by a method of engineering an AAV capsid to target the CNS, comprising: a) identifying an amino acid sequence encoding a peptide fragment that specifically binds to a GPI-anchored BBB ligand; and b) modifying an AAV HVRVIII site to express the amino acid sequence, wherein the engineered capsid binds to the GPI-anchored BBB ligand.

[0012] In one embodiment, a method for detectably labeling a CNS target is provided herein, comprising administering an AAV capsid that binds to a GPI-anchored ligand on the BBB and is conjugated to a detectable effector entity, wherein the AAV capsid, upon binding to the GPI-anchored BBB ligand, transports the detectable effector entity conjugated to it across the BBB. In one embodiment, the ligand is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRINI, GFRA3, GPIHBP1, MACF1, and SEC24B. In yet another embodiment, the detectable effector entity comprises a peptide, a nucleic acid, an siRNA, an antibody, an antibody fragment, a small molecule, a lipid nanoparticle, or a cytotoxic agent.

[0013] Other aspects and advantages of these compositions and methods are further described in the detailed description that follows. [Brief description of the drawings]

[0014] [Figure 1A-1B]We show that the ability of PHP.B to cross the BBB in mice is strain specific and inherited as a codominant trait. (Figure 1A) Representative direct GFP fluorescence in the brain of inbred and outbred strains injected IV with 1 x 1012 gc of AAV-PHP.B.CB7.EGFP. The percentage of F2 mice showing intermediate (55.5%), minimal (27.8%) and strong (16.7%) intracerebral transduction suggests Mendelian inheritance. A total of 18 F2 mice were injected. Scale bar 100 μm. (Figure 1B) Vector genome copies measured by Taqman qPCR in the brain (by mean and error bars (standard deviation) per group). Analysis of variance, Kruskal-Wallis test followed by Dunn's multiple comparison test: *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. [Figures 2A-2D]We show that the Ly6a gene, located on chromosome 15, is associated with a high brain transduction phenotype. (Figure 2A) Overview of the WES association study design. F1s were generated by crossing inbred strains C57BL / 6J and BALB / cJ, all heterozygous with an intermediate brain transduction phenotype. F2s were generated by crossing F1s together, producing a range of phenotypes and genotypes. WES libraries were generated from genomic DNA isolated from 16 mice (4 F1 and 12 F2) and mapped to the reference genome (GRCm38, C57BL / 6J). (Figure 2B) Manhattan plot showing genetic variants significantly associated (red line, p ≤ 5E-8) with BBB-wide PHP.B transduction in mice. The strongest association is observed within the D3 karyotype band (green), which contains two labeled missense mutations in the Ly6a gene. Genetic confirmation that the presence of top candidate Ly6a (Sca-1) is required for intracerebral transduction of AAV-PHP.B across the BBB in vivo. (Figure 2C) Representative direct GFP fluorescence in the brain (hippocampus) and liver of inbred WT and Ly6a-null mice after IV administration of 1x1012 gc of AAV-PHP.B.CB7.EGFP. (Figure 2D) LY6A immunostaining in the brain cortex of KO and WT mice using a rat monoclonal antibody that recognizes both haplotypes (clone D7). LY6A expression is strong in brain capillaries of C57BL / 6J WT mice, weak in brain capillaries of BALB / cJ WT mice, and undetectable in the brain of all KO mice. Scale bars: (Figure 2C) 100 μm, (Figure 2D) 50 μm. [Figure 3A-3B] Figure 3 shows that the Ly6a haplotype determines the ability of AAV-PHP.B to cross the BBB in mice. (Figure 3A) Ly6a haplotypes from a selection of inbred mouse strains (data according to the Mouse Genome Project, MM10 dbSNP142). (Figure 3B) AAV-PHP.B intracerebral transduction after systemic administration in inbred mice carrying the reference Ly6a gene (Ly6b haplotype, C57BL / 6J-like) and in strains carrying a BALB / cJ-like SNP (Ly6a haplotype). Scale bar 100 μm. [Figure 4A-C]AAV-PHP.B binds to LY6A protein. (FIG. 4A) ELISA measurements of AAV-PHP.B, AAV-PHPeB, AAV9, or the inactive AAV-PHP.B V592G mutant binding to LY6A protein. Both AAV-PHPeB and PHP.B bind to LY6A, with a higher maximum signal for the C57BL / 6J variant. AAV9 and AAV-PHP.B V592G do not bind to LY6A. Curves show the average of three replicates (error bars are standard error of the mean). (FIG. 4B) AAV.LacZ transduction assay in Ly6a transfected cells. Transient expression of both Ly6a haplotypes in HEK293 cells enhances AAV-PHP.B but not AAV9 transduction, while another ly6 family member, ly6c1, has no effect. AAV9- and AAV-PHP.B-mediated transduction efficiencies within the same transfection conditions are compared using two-way ANOVA followed by mean comparison tests using Tukey's multiple comparison test (GraphPAD Prism). (Figure 4C) AAV.LacZ transduction assay in the presence of anti-LY6A antibody. Enhanced transduction (MOI 10,000) is prevented by preincubation with rat monoclonal anti-LY6A antibody (clone D7, 100 nM, 1 h at +4 °C). Transduction efficiencies in the presence of different antibodies are compared within the same transfection conditions using two-way ANOVA followed by mean comparison tests using Tukey's multiple comparison test (GraphPAD Prism). Bar graphs showing the mean of 6 replicates (Figure 4B) or 8 replicates (Figure 4C) (error bars are standard deviation). [Figure 5A-5B] In BALB / cJ mice, AAV-PHP.B cannot cross the BBB but can transduce the brain after intracerebroventricular administration. Direct GFP fluorescence in the brain after intravenous (IV, 1x1012 gc) (Figure 5A) or intracerebroventricular (ICV, 1x1011 gc) (Figure 5B) administration to C57BL / 6J and BALB / cJ mice. Scale bars: (Figure 5A) 1 mm, (Figure 5B) 100 μm. [Figure 6]Mutations in the operational domain of AAV-PHP.B are shown to abrogate its ability to cross the BBB. Direct GFP fluorescence in liver and brain after IV administration of 1x1012gc AAV-PHP.B-V592G in C57BL / 6J mice. Scale bar 100μm. [Figure 7] AAV-PHP.eB-mediated intracerebral transduction is shown to be strain dependent. Direct GFP fluorescence in the brain following intravenous (IV) administration of 1x1012gc of AAV-PHP.eB.CB7.EGFP to C57BL6 / J and BALBc / J mice. AAV-PHP.eB does not cross the BBB in BALB / cJ. Scale bar 100 μm. [Figure 8] Figure 1 shows that PHP.B variants have varying affinities for the Ly6a receptor. Valine 592 of the AAV9-PHP.B capsid, located within the 588-TLAVPFK peptide insert, was subjected to saturation mutagenesis. Each vector variant was purified and individually tested for binding affinity to Ly6a using SPR. From left to right: Biacore sensorgrams for high-affinity, native-affinity, and low-affinity variants of AAV9-PHP.B. [Figure 9] Figure 1 shows the effect of Ly6a binding affinity on cell transduction. The transduction efficiency of each affinity variant was quantified by expression of β-galactosidase in HEK293 cells stably expressing Ly6a. The low affinity variant shows a modest increase (2-fold) in transduction efficiency compared to the high affinity variant at low MOI. Any level of binding to the Ly6a receptor is sufficient to improve transduction efficiency by more than 10-fold compared to AAV9. [Figure 10A-10B]The effect of Ly6a binding affinity on biodistribution is shown. (Figure 10A) Vectors with affinity for the Ly6a receptor show comparable levels of localization to brain tissue. When biodistribution was tracked by liver expression of eGFP, variants with low affinity for Ly6a show increased localization to and expression in liver tissue. (Figure 10B) Brain histology shows that high and low affinity vectors have similar biodistribution in brain tissue, but vectors with low to moderate affinity for Ly6a show improved expression. [Figure 11] The effect of PHP.B peptide presentation on valency and cell transduction is shown. Chimeric capsids were produced by altering the ratio of plasmids encoding either the AAV9 capsid gene or the AAV9-PHP.B capsid gene used during vector production. The transduction efficiency of each chimeric variant was quantified by expression of β-galactosidase in HEK293 cells stably expressing Ly6a. [Figure 12] 1 shows multivalent display of the Ly6a domain required for binding between soluble Ly6a and PHP.B. [Figure 13] Shown is staining of brain sections from non-human primates for the target molecule SEMA7A, which revealed high density expression on brain endothelium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] As described herein, the inventors have identified a ligand on the BBB that mediates efficient transport of AAV vectors to the CNS. Although the ligand, LY6A (Sca-1), was previously known, the functional role of this GPI-anchored protein was thought to be limited to hematopoietic biology. Thus, the inventors have shown, for the first time, that GPI-anchored proteins may play an important role in facilitating the delivery of viral vectors across the BBB. Thus, novel compositions and methods are disclosed herein for targeting GPI-anchored ligands of the BBB to deliver gene therapy vectors and / or therapeutic agents to the CNS.

[0016] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art of biology, biotechnology, and molecular biology, and by reference to published documents which provide general guidance to those of ordinary skill in the art for many of the terms used in this application. The definitions herein are provided for clarity only and are not intended to limit the claimed invention.

[0017] As used herein, the term "subject" refers to a mammal, including humans, veterinary or agricultural animals, domestic or pet animals, and animals typically used in clinical research. In one embodiment, the subject of these methods and compositions is a human. Still other suitable subjects include, but are not limited to, mice, rats, dogs, cats, pigs, cows, sheep, non-human primates, and others. As used herein, the term "subject" is used interchangeably with "patient."

[0018] "Blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, which is formed by tight junctions in the plasma membrane of the brain capillary endothelium, creating a tight barrier that limits the transport of molecules, even very small molecules such as urea (60 daltons), into the brain. The BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retina barrier in the retina are continuous capillary barriers in the CNS, and are collectively referred to herein as the blood-brain barrier or BBB. The BBB also encompasses the blood-CSF barrier (choroid plexus), where the barrier is composed of ependymal cells rather than capillary endothelial cells.

[0019] "Central nervous system" or "CNS" refers to the complex of nervous tissue that controls bodily functions, including the brain and spinal cord.

[0020] As used herein, "systemic delivery" refers to delivery that results in widespread biodistribution of the compositions described herein within an organism, for example as a result of such compositions moving from one location to another via the systemic circulation. Systemic delivery means that a useful, preferably therapeutic amount of the composition is exposed to most parts of the body. Systemic delivery of the compositions provided herein is preferably achieved by intravenous delivery, but can also be achieved by other delivery routes, including oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, and other parenteral routes.

[0021] As used herein, "neurological disorder" refers to a disease or disorder that affects and / or has a cause in the CNS.Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorder, and lysosomal storage disease. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, and multiple system atrophy), striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsies, motor neuron diseases, and heterogeneous degenerative disorders of the nervous system (including, but not limited to, Canavan disease, Huntington's disease, and neuronal ceroid lipofuscinosis). , Alexander disease, Tourette's syndrome, Menkes kinky hair syndrome, Cocaine syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementias (including but not limited to Pick's disease and spinocerebellar ataxia), epidural gliomas, and cancers affecting the CNS (including but not limited to glioma, glioblastoma multiforme, meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma, medulloblastoma, ganglioglioma, schwannoma, neurofibroma, neuroblastoma, and epidural, intramedullary or intradural tumors).

[0022] Viral or microbial infections of the CNS include, but are not limited to, viruses (i.e., influenza, HIV, poliovirus, rubella), bacteria (i.e., Neisseria species, Streptococcus species, Pseudomonas species, Proteus species, E. coli, S. aureus, Pneumococcus species, Meningococcus species, Haemophilus species, and Mycobacterium tuberculosis), and other microorganisms such as fungi (i.e., yeast, Cryptococcus neoformans), parasites (i.e., toxoplasma gondii), or amoebas that result in CNS pathophysiology, including, but not limited to, meningitis, encephalitis, myelitis, vasculitis, and abscesses, which may be acute or chronic.

[0023] As used herein, an "imaging agent" is a compound that has one or more properties that allow its presence and / or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds that incorporate labeled entities that allow for detection. A "detectable label" is a marker used for detection or imaging. Examples of such labels include radiolabels, fluorophores, chromophores, or affinity tags. In one embodiment, the label is a radiolabel used in medical imaging, such as technetium-99m or iodine-123 for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), or a spin label (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc.).

[0024] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction.Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, Pb-212, and radioisotopes of Lu), chemotherapy drugs or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents), growth inhibitors, enzymes and their fragments (e.g., nucleases), antibiotics, toxins (including small molecule toxins, or fragments and / or variants thereof, such as enzymatically active toxins of bacterial, fungal, plant, or animal origin).

[0025] As used herein, the term "treatment" and its variants such as "treat" or "treating" refer to clinical intervention in an attempt to change the natural course of the individual being treated, and may be performed for prophylaxis or during the clinical pathological course. Desirable effects of treatment include, but are not limited to, prevention or reduction of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement or alleviation of the disease state, and remission or improvement of prognosis. In some embodiments, the effectors described herein are used to delay the onset of disease or to slow the progression of disease. Similarly, "treating cancer" may be described by several different parameters, including, but not limited to, reduction of tumor size in an animal with cancer, reduction of tumor growth or proliferation in an animal with cancer, prevention, inhibition or reduction of the extent of metastasis, and / or prolongation of survival of an animal with cancer compared to a control.

[0026] As used in connection with the methods and compositions described herein, the term "antibody" refers to an intact immunoglobulin having two light chains and two heavy chains or fragments thereof that can bind to a biomarker protein or a fragment of a biomarker protein. Thus, a single isolated antibody or antigen-binding fragment thereof can be a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, or a bispecific antibody or multispecific construct that can bind to two or more antigens. As used herein, the term "antibody" can also refer to an antibody fragment.

[0027] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody (such variants are generally present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are not contaminated by other immunoglobulins. The modifier "monoclonal" denotes the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or can be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can be isolated from phage antibody libraries, for example, using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991). Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies, including antigen-binding fragments thereof. Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins), in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780).

[0028] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will contain substantially all of at least one (typically two) variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of human immunoglobulin sequences, except for the FR substitution(s) noted above. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region, typically a human immunoglobulin constant region. For further details, see Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol 2:593-596 (1992).

[0029] The term "antibody fragment" as used in connection with the methods and compositions described herein refers to a structure smaller than an intact antibody that has antigen-binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (such as single-chain Fab, scFv, and multispecific antibodies formed from antibody fragments). "Single-chain Fab" formats are described, for example, in Hust M.et al.BMC Biotechnol.2007 Mar8;7:14. scFV constructs include complementary scFvs produced as single chains (tandem scFv) or bispecific tandem scFv.

[0030] As used herein, "specifically binding," "binds specifically to," and "specific binding" refer to, for example, an antibody that selectively or preferentially binds to an antigen. For example, with respect to the antibodies or capsids and methods of use thereof described herein, "specific binding" refers to selective binding, which refers to the ability of an antibody or capsid to bind to one or more epitopes of an antigen of interest or a binding partner without substantially recognizing and binding to other molecules in a sample or environment containing a mixed population of antigens. Specific binding interactions are mediated by one, or typically more, non-covalent bonds between the binding molecules or binding partners. In certain aspects described herein, the binding partner is a capsid protein (or a portion thereof) or a molecule (such as an antibody) conjugated to a capsid protein that specifically binds to a target ligand (i.e., a GPI-anchored protein on the BBB). In one embodiment, the targeted GPI-anchored protein is a member of the Ly6A family. In certain embodiments, the GPI-anchored protein is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

[0031] Various embodiments herein are presented using the word "comprising" to include other components or method steps. When "comprising" is used, it should be understood that the relevant embodiment includes descriptions using the term "consisting of," which excludes other components or method steps, and "consisting essentially of," which excludes any components or method steps that materially change the nature of the embodiment or invention.

[0032] It is to be understood that the terms "a" or "an" refer to one or more, e.g., "a ligand" refers to one or more ligands. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0033] As used herein, the term "about," when referring to a measurable value, such as an amount, temporal duration, and the like, is meant to encompass up to ±10% variation from the specified value, where such variation is appropriate for carrying out the disclosed method. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (e.g., molecular weight, reaction conditions, and the like) used in the specification and claims should be understood to be modified in all instances by the term "about."

[0034] As used herein, the terms "compound," "composition," or "agent" may be used interchangeably to discuss a therapeutic composition.

[0035] With respect to the description of the invention provided herein, each of the compositions described is contemplated, in separate embodiments, to be useful in the methods of the invention. In addition, each of the compositions described herein that are useful in the methods is also contemplated, in separate embodiments, to be an embodiment of the invention itself.

[0036] In one aspect, a recombinant AAV is provided that has a capsid conjugated with an effector entity.The AAV capsid described herein has a binding partner that specifically binds to the GPI anchor ligand on the BBB to facilitate the delivery of the conjugate that comprises the effector entity to the CNS.In one embodiment, the GPI anchor ligand is Ly6E. Additional conjugates provided herein include AAV capsids that specifically bind to other GPI-anchored proteins on the BBB, including, but not limited to, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

[0037] The term "AAV" as used herein refers to dozens of naturally occurring and available adeno-associated viruses, as well as artificial AAVs. Adeno-associated virus (AAV) viral vectors are AAV DNase-resistant particles with AAV protein capsids, into which nucleic acid sequences are packaged for delivery to target cells. AAV capsids consist of 60 capsid protein subunits VP1, VP2, and VP3, arranged in icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending on the AAV selected. Various AAVs may be selected as the source of capsids for the above-mentioned AAV viral vectors. See, for example, U.S. Published Patent Application No. 2007 / 0036760-A1, U.S. Published Patent Application No. 2009 / 0197338-A1, and EP1310571. See also WO2003 / 042397 (AAV7 and other simian AAVs), U.S. Patent No. 7,790,449 and U.S. Patent No. 7,282,199 (AAV8), WO2005 / 033321 and U.S. Patent No. 7,906,111 (AAV9), and WO2006 / 110689, WO2003 / 042397 (rh10), and WO2018 / 160582 (AAVhu68). These documents also describe other AAVs that can be selected to generate AAVs (incorporated by reference). Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein can be readily selected from among any AAV, including, but not limited to, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVAnc80, AAVrhlO, and AAVPHP.B, as well as any variant of the known or mentioned AAVs, or any yet to be discovered AAV or variant, or mixtures thereof. See, e.g., WO2005 / 033321 (hereby incorporated by reference).In one embodiment, the AAV capsid is an AAV1 capsid or a variant thereof, an AAV8 capsid or a variant thereof, an AAV9 capsid or a variant thereof, an AAVrh.10 capsid or a variant thereof, an AAVrh64R1 capsid or a variant thereof, an AAVhu.37 capsid or a variant thereof, or an AAV3B or a variant thereof.

[0038] The amino acid sequence of the AAVPHP.B variant capsid is publicly available (GenBank accession number KU056473) and is as follows: MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 1)

[0039] The AAVPHP.eB variant capsid was described by Chan et al. (Nat. Neurosci., Aug. 2017, 20(8):1172-9). The amino acid sequence is publicly available (GenBank accession number MF187357.1) and is as follows: (SEQ ID NO:2)

[0040] AAV capsids contain nine hypervariable regions (HVRs) that show the most sequence variance across AAV isolates. See Govindasamy et al, J Virol. 2006 Dec;80(23):11556-70. Epub 2006 Sep13, incorporated herein by reference. For example, AAV9 VP differs in nine variable surface regions (VR-I-IX) compared to AAV4, but only in three variable surface regions (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. See, e.g., DiMattia et al. J Virol. 2012 Jun;86(12):6947-58, incorporated herein by reference. In one embodiment, the AAV capsid has one or more mutations in an HVR, such as HVRVIII. In certain embodiments, the mutations in the HVRVIII region confer specific binding (i.e., form a binding partner) to a GPI-anchored protein on the BBB. Thus, the AAV capsid protein specifically binds to a GPI-anchored ligand on the BBB to modify its infectivity or facilitate the delivery of an effector entity conjugated thereto. It can be operated to advance

[0041] In one embodiment, the above-mentioned method is used to generate a binding partner to provide a modified AAV capsid, or an AAV capsid linked to other molecules (e.g., antibodies) (i.e., the capsid specifically binds to a GPI-anchored ligand on the BBB via an intermediate). Thus, the AAV capsid of the conjugate described herein can be directly bound to a ligand or can be conjugated to an intermediate (e.g., an antibody or an oligonucleotide) to form a binding partner of the ligand. Methods that can be used to generate AAV capsids with modified receptor binding and / or introduce receptor binding partners are known in the art and are provided, for example, in the following references (incorporated herein by reference): Munch et al.Molecular Therapy,2013 Jan;21(1):109-18, Ried et al.J Virol.2002 May;76(9):4559-66, Ponnazhagan et al.J Virol.2002 Dec;76(24):12900-07, and Katrekar D et al.Sci Rep.2018 Feb 26;8(1):3589.

[0042] As used herein, "conjugate" or "conjugated" refers to, for example, an AAV capsid bound to one or more effector entities. In certain embodiments, the AAV capsid and the effector entity are linked by a linker. As used herein, "linker" refers to a chemical linker or a single-chain peptide linker that covalently links the AAV capsid and the effector entity of the conjugate described herein. The covalent bond can be either direct or via a linker. In certain embodiments, direct conjugation is by the formation of a covalent bond between a reactive group on the capsid and a corresponding group or acceptor (e.g., on the neurological agent). In certain embodiments, direct conjugation is performed by modifying (e.g., genetically modifying) one of the two molecules to be conjugated to include a reactive group (for example, a sulfhydryl group or a carboxyl group) that forms a covalent bond to the other molecule to be conjugated under appropriate conditions. As one non-limiting example, a molecule (e.g., an amino acid) with a desired reactive group (e.g., a cysteine ​​residue) can be introduced into the capsid, for example, to form a disulfide bond with a neurological agent. Methods for covalently conjugating nucleic acids with proteins are also known in the art (e.g., photocrosslinking, see, e.g., Zatsepin et al. Russ. Chem. Rev. 74:77-95 (2005)). Non-covalent conjugation can be any non-covalent means, including hydrophobic bonds, ionic bonds, electrostatic interactions, etc., as can be easily understood by those skilled in the art.

[0043] Conjugation can also be performed using various linkers. For example, antibodies and neuropharmaceuticals can be conjugated using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-L-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl H adipimidate), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-D TPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026. Peptide linkers consisting of 1-20 amino acids linked by peptide bonds may also be used. In certain such embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other such embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker may be a "cleavable linker" that facilitates release of the neurological agent upon delivery to the brain. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0044] Conjugates may include conjugates prepared with cross-linking reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0045] As used herein, a "CNS antigen" or "CNS target" is an antigen and / or molecule expressed in the CNS, including the brain, that can be targeted with an effector entity, such as an antibody or small molecule. Examples of such antigens and / or molecules include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.

[0046] In certain embodiments, the compositions described herein are useful for the treatment of Alzheimer's disease and include adumanucab (Biogen), bapineuzumab (Elan, a humanized mAb directed against the amino terminus of Aβ), solanezumab (Eli Lilly, a humanized mAb against the central portion of soluble Aβ), gantenerumab (Chugai and Hoffmann-La Roche, a fully human mAb directed against both the amino terminus and the central portion of Aβ), crenezumab (Genentech, a humanized mAb acting on monomeric and conformational epitopes, including oligomeric and protofibrillar forms of Aβ), BAN2401 (Esai), and other human mAbs that are directed against Aβ. Co., Ltd., a humanized immunoglobulin G1 (IgG1) mAb that selectively binds to Aβ protofibrils, believed to promote the clearance of Aβ protofibrils and / or neutralize their toxic effects on neurons in the brain), GSK933776 (a humanized IgG1 monoclonal antibody directed to the amino terminus of Aβ), AAB-001, AAB-002, AAB-003 (Fc-engineered bapineuzumab), SAR228810 (a humanized mAb directed to protofibrils and low molecular weight Aβ), BIIB037 / BART (fully human IgG1 against insoluble fibrillar human Aβ, Biogen Idec), and an anti-Aβ antibody such as m266 (relative specificity for Aβ oligomers) [Brody and Holtzman, Annu Rev Neurosci, 2008;31:175-193].

[0047] In another embodiment, the compositions described herein comprise antibodies useful for treating Parkinson's disease, e.g., leucine-rich repeat kinase 2, daldarin (LRRK2), synukin (SYN) and / or leucine-rich repeat kinase 2 (LRRK3). Antibodies include those directed against leukemia, alpha-synuclein, or DJ-1 (PARK7). Other antibodies may include PRX002 (Prothena and Roche) Parkinson's disease and related synucleinopathies. These antibodies, particularly anti-synuclein antibodies, may also be useful in treating one or more lysosomal storage diseases.

[0048] In yet another embodiment, the compositions described herein comprise antibodies useful for the treatment of multiple sclerosis, including natalizumab (humanized anti-a4-Inguin, iNATA, Tysabri, Biogen Idec and Elan Pharmaceuticals, approved in 2006), alemtuzumab (Campus-1H, humanized anti-CD52), rituximab (Ritujin, chimeric anti-CD20), daclizumab (Zenepax, humanized anti-CD25), ocrelizumab (humanized, anti-CD20, Roche), ustekinumab (CNTO-1275, human anti-IL12p40+IL23p40), anti-LINGO-1 and ch5D12 (chimeric anti-CD40), and rHIgM22 (remyelinating monoclonal antibody, Acorda and Foundation for Medical Education and Research), etc. Still other anti-a4-integrin antibodies, anti-CD20 antibodies, anti-CD52 antibodies, anti-IL17 antibodies, anti-CD19 antibodies, anti-SEMA4D antibodies, and anti-CD40 antibodies may be delivered via the conjugates described herein.

[0049] In one embodiment, the compositions provided herein include antibodies useful for treating ALS, such as antibodies against the enzyme superoxide dismutase 1 (SOD1) and variants thereof (e.g., ALS variant G93A, C4F6 SOD1 antibody), MS785 (directed to the darling-1 binding region), and antibodies against neurite outgrowth inhibitors (NOGO-A or reticulon 4), such as GSK1223249, ozanezumab (humanized, GSK, also described as useful for multiple sclerosis).

[0050] Methods for generating AAV capsids, coding sequences, and thus producing rAAV viral vectors have been described.See, for example, Gao, et al, Proc.Natl.Acad.Sci.USA100(10), 6081-6086(2003), and US2015 / 0315612.In one embodiment, the AAV that supplies the capsid is AAV1 or a variant thereof.In another embodiment, the AAV that supplies the capsid is AAV3B or a variant thereof.In one embodiment, the AAV that supplies the capsid is AAV9 or a variant thereof.In yet another embodiment, the AAV that supplies the capsid is AAV of clade E or a variant thereof. Such AAVs include rh.2, rh.10, rh.25, bb.1, bb.2, pi.1, pi.2, pi.3, rh.38, rh.40, rh.43, rh.49, rh.50, rh.51, rh.52, rh.53, rh.57, rh.58, rh.61, rh.64, hu.6, hu.17, hu.37, hu.39, hu.40, hu.41, hu.42, hu.66, and hu.67. This clade further includes modified rh.2, modified rh.58, and modified rh.64. See WO2005 / 033321, which is incorporated herein by reference.

[0051] As used herein, the term "variant" with respect to AAV refers to any AAV sequence derived from a known AAV sequence, including sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more sequence identity across amino acid or nucleic acid sequences. In another embodiment, the AAV capsid includes a variant that may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid provided herein and / or known in the art. In one embodiment, the AA The V capsid shares at least 95% identity with the AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made across any of the variable proteins (e.g., vp1, vp2, or vp3). In another embodiment, a self-complementary AAV is used.

[0052] As used herein, "artificial AAV" refers to, but is not limited to, an AAV with a non-natural capsid protein. Such an artificial capsid can be produced by any suitable technique using a selected AAV sequence (e.g., a fragment of vp1 capsid protein) in combination with a heterologous sequence (which can be obtained from a different selected AAV, a non-contiguous part of the same AAV, a non-AAV viral source, or a non-viral source). An artificial AAV can be, but is not limited to, a pseudotyped AAV capsid, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. A pseudotyped vector is useful in the present invention, in which the vector genome packaged within the AAV capsid has inverted terminal repeats (ITRs) from a source different from the capsid. In one embodiment, AAV2 / 5 and AAV2 / 8, which have ITRs from AAV2 and capsids from AAV5 and AAV8, respectively, are exemplary pseudotyped vectors. The selected genetic element can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. The methods used to create such constructs are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0053] In one embodiment, the AAV capsid that forms a conjugate with the effector entity is an "empty" capsid. Such an empty capsid may contain no detectable genomic sequence of the expression cassette or may contain only partially packaged genomic sequence that is insufficient to achieve expression of the gene product.

[0054] In yet another embodiment, the AAV capsid provided comprises a vector genome having an expression cassette comprising a nucleic acid sequence encoding a heterologous gene. As used herein, "vector genome" refers to a nucleic acid sequence packaged into the AAV capsid that forms the viral particle. Such a nucleic acid sequence comprises an AAV inverted terminal repeat (ITR). The vector genome comprises at least, from 5' to 3', the 5'ITR of AAV2, the heterologous coding sequence, and the 3'ITR of AAV2. However, ITRs may be selected from different sources of AAV other than AAV2. Additionally, other ITRs may be used. Additionally, the vector genome preferably comprises a control sequence that induces the expression of a gene of interest. In one embodiment, it is preferred to use a tissue-specific promoter suitable for the expression of a heterologous gene or expression cassette delivered to the CNS.

[0055] When used to describe a nucleic acid sequence or protein, the term "heterologous" means that the nucleic acid or protein originates from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. When used in reference to a protein or a nucleic acid of a plasmid, expression cassette, or vector, the term "heterologous" indicates that there is another sequence or subsequence of the protein or nucleic acid such that the protein or nucleic acid is not found in the same relationship to each other in nature. Thus, in one aspect, the AAV capsids provided herein contain a sequence encoding at least one gene that may be therapeutically beneficial, particularly when expressed in the CNS.

[0056] An "effector entity" is a molecule that crosses the BBB and transports the Effector entities refer to molecules that are transported to the brain. Effector entities typically have a characteristic therapeutic activity that is desired to be delivered to the brain. Effector entities include drugs and cytotoxic agents for treating neurological disorders, and include peptides, proteins, nucleic acids (e.g., siRNAs), antibodies (particularly monoclonal antibodies or fragments thereof), small molecules, and lipid nanoparticles that can be targeted to brain targets. In certain embodiments, effector entities can include detectable labels that are useful for imaging diagnostics of the CNS.

[0057] In one embodiment, the AAV capsid is conjugated to a neuropathic drug, chemotherapeutic agent, and / or imaging agent to transport the drug, chemotherapeutic agent, and / or imaging agent across the BBB.

[0058] For neuropathic disorders, non-limiting examples include narcotic / opioid analgesics (i.e., morphine, fentanyl, hydrocodone, meperidine, methadone, oxymorphone, pentazocine, propoxyphene, tramadol, codeine, and oxycodone), nonsteroidal anti-inflammatory drugs (NSAIDs) (i.e., ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketorolac, , mefenamic acid, meloxicam, nabumetone, oxaprozin, piroxicam, sulindac, and tolmetin), corticosteroids (i.e., cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone, and triamcinolone), antimigraine medications (i.e., sumatriptan, almotriptan, frovatriptan, sumatriptan, rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan, and ergotamine), acetaminophen, salicylates (i.e., aspirin, choline salicylate, magnesium salicylate, diflunisal, and salsalate), anticonvulsants (i.e., carbamazepine, clonazepam, gabapentin, lamotrigine, pregabalin, tiagabine, and topiramate), anesthetics (i.e., isoflurane, trichloroethylene, halothane, sevoflurane, benzocaine, chloroprocaine, cocaine, cyclomethicine, dimethicone, Analgesic neuropathic drugs may be selected, including analgesic neuropathic drugs, such as cerebroside, propoxycaine, procaine, novocaine, proparacaine, tetracaine, articaine, bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine, saxitoxin, and tetrodotoxin, and COX-2 inhibitors (i.e., celecoxib, rofecoxib, and valdecoxib). In the case of neuropathic disorders involving vertigo, anti-vertigo neuropathic drugs may be selected, including, but not limited to, meclizine, diphenhydramine, promethazine, and diazepam.For neuropathic disorders involving nausea, antiemetic neuropharmaceuticals may be selected, including, but not limited to, promethazine, chlorpromazine, prochlorperazine, trimethobenzamide, and metoclopramide. For neurodegenerative diseases, growth hormones or neurotrophic factors may be selected, including, but not limited to, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4 / 5, fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliary nerve. These include trophic factor (CNTF), glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin, persephin, interleukin, glial cell line-derived neurotrophic factor (GFR), granulocyte colony-stimulating factor (CSF), granulocyte-macrophage colony-stimulating factor, netrin, cardiotrophin-1, hedgehog, leukemia inhibitory factor (LIF), midkine, pleiotrophin, bone morphogenetic protein (BMP), netrin, saposin, semaphorin and stem cell factor (SCF).

[0059] In the case of cancer, the neurological agent can be a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents (such as thiotepa and CYTOXAN®, cyclosphosphamide, etc.); alkylsulfonates (such as busulfan, improsulfan, and piposulfan), aziridines (such as benzodopa, carboquone, meturedopa, and uredopa), ethylenimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine), acetophenone, ... togenins (especially bullatasin and bulltacinone), delta-9-tetrahydrocannabinol (dronabinol, MARINOL®), β-lapacol, lapachol, colchicine, betulinic acid, camptothecins (including the synthetic analog topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin), bryostatin, kallistatin, CC -1065 (including synthetic analogs of adrestin, carzelesin, and bizelesin), podophylpodoxin, podophyllic acid, teniposide, cryptophycins (especially cryptophycin 1 and cryptophycin 8), dolastatins, duocarmycins (including synthetic analogs KW-2189 and CB1-TM1), erytherobin, pancratistatin, sarcodictin, spongiostatin, nitrogen mustards (chlorambucil, chlornaphazine, chlorophosphamide, , estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.), nitrosoureas (carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, etc.), antibiotics such as enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin γ1I and calicheamicin ω1I (see Agnew, Chem Intl. Ed. Engl, 33:183-186 (1994)), dynemicins, including dynemicin A, esperamicin, neocarzinostatin chromophores, and related chromoprotein enediyne antibiotic chromophores),Aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcetin, Mitomycins such as thromycin and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodolubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)), folic acid analogues (such as denopterin, methotrexate, pteropterin, trimetrexate), purine analogues (fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.), pyrimidine analogues (ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.), androgens (calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.), antiadrenal (aminoglutethimide, mitotane, trilostane, etc.), folic acid supplements (floric acid, etc.), aceglatone, aldophosphamide glycosides, aminolevulinic acid, enyluracil , amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids (such as maytansine and ansamitocin), mitoguazone, mitoxantrone, mopidamol, nitraelin, pentostatin, phenamet, pirarubicin, rosoxantrone, 2-ethylhydrazide, procarbazine, PSK (registered trademark), polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane,Rhizoxin, Schizophrenia, Filan, spirogermanium, tenuazonic acid, triazicon, 2,2',2''-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veracrine A, and anguidin), urethanes, vindesine (ELDISINE®, FILDESIN®), dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), thiotepa, taxoids (e.g., TAXOL®, paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ)), ABRAXANETM cremophor-free, albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTEPvE®, docetaxel (Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine (GEMZAR®), 6-thioguanine, mercaptopurine, methotrexate, platinum analogues (such as cisplatin and carboplatin), vinblastine (VELBAN®), platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine (ONCOVIN®), oxaliplatin, leucovorin, vinorelbine (NAVELBINE®), novantrone, edatrexate, daunomycin, aminopterin, These include ibandronate, the topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO) retinoids (such as retinoic acid), capecitabine (XELODA®), pharmaceutically acceptable salts, acids, or derivatives of any of the above, and combinations of two or more of the above, such as CHOP (short for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (short for the treatment regimen of oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™)).

[0060] Chemotherapeutic agents are also useful in certain aspects of the invention described herein, acting to modulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth, often as a systemic or form of whole-body treatment. They can also be hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, and FARESTON® toremifene, antiprogesterones, estrogen receptor downregulators (ERDs), agents that function to suppress or shut down the ovaries (e.g., luteinizing agents such as LUPRON® and ELIGARD® leuprolide acetate), and the like. LHRH agonists, goserelin acetate, buserelin acetate, and tripterelin), other antiandrogens (such as flutamide, nilutamide, bicalutamide), and aromatase inhibitors that block aromatase, an enzyme that regulates estrogen production in the adrenal glands (e.g., 4(5)-imidazole, aminoglutethimide, MEGASE® - megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, ARIMIDEX® anastrozole).Additionally, such definition of chemotherapeutic agent includes bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®, DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid / zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate), as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog), antisense oligonucleotides (particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell growth, e.g., Such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R), vaccines (such as the THERATOOPE® vaccine) and gene therapy vaccines (e.g., the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine, and the VAXID® vaccine), LURTOTECAN® topoisomerase 1 inhibitors, ABARELLX® rmRH, lapatinib tosylate (a dual tyrosine kinase small molecule inhibitor of ErbB-2 and EGFR, also known as GW572016), and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Another group of compounds that may be selected as neuropharmaceuticals for the treatment or prevention of cancer are anti-cancer immunoglobulins (including but not limited to trastuzumab, bevacizumab, alemtuzumab, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, panitumumab and rituximab). In some cases, antibodies, along with toxic labels, may be used to target and kill the desired cells (i.e., cancer cells) (including but not limited to tositumomab with a radiolabel).

[0061] For lysosomal storage diseases, the enzyme itself that is impaired in the disease or else a neuropharmaceutical that mimics the activity of the enzyme may be selected. Exemplary recombinant enzymes for the treatment of lysosomal storage diseases include, but are not limited to, those described in, for example, U.S. Patent Application Publication No. 2005 / 0142141 (i.e., α-L-iduronidase, iduronate-2-sulfatase, N-sulfatase, α-N-acetylglucosaminidase, N-acetylgalactosamine-6-sulfatase, β-galactosidase, arylsulfatase B, β-glucuronidase, acid α-glucosidase, glucocerebrosidase, α-galactosidase A, hexosaminidase A, acid sphingomyelinase, β-galactocerebrosidase, β-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase, palmitoyl protein-thioesterase 1, and tripeptidyl aminopeptidase 1).

[0062] In the case of amyloidosis, the following neurological drugs may be selected, including but not limited to, antibodies or other binding molecules (including but not limited to small molecules, peptides, aptamers, or other protein binders) that specifically bind to a target (selected from beta-secretase, tau, presenilin, amyloid precursor protein or a portion thereof, amyloid beta peptide or an oligomer or fibril thereof, death receptor 6 (DR6), receptor for advanced glycation end products (RAGE), parkin, and huntingtin), cholinesterase inhibitors (i.e., galantamine, donepezil, rivastigmine, and tacrine), NMDA receptor antagonists (i.e., memantine), monoamine depleting agents (i.e., tetrabenazine), ergoloid mesylates, anticholinergic antiparkinsonian agents (i.e., procyclidine, diphenhydramine, trihedramine, cyclosporine ... cyclosporine, ...

[0063] In the case of viral or microbial diseases, neurological agents may be selected from, but are not limited to, antiviral compounds (adamantane antivirals (i.e., rimantadine and amantadine), antiviral interferons (i.e., PEG-interferon alpha-2b), chemokine receptor antagonists (i.e., maraviroc), integrase inhibitors (i.e., integrase inhibitors), antiviral agents ... Zeta-transfer inhibitors (i.e., raltegravir), neuraminidase inhibitors (i.e., oseltamivir and zanamivir), non-nucleoside reverse transcriptase inhibitors (i.e., efavirenz, etravirine, delavirdine and nevirapine), nucleoside reverse transcriptase inhibitors (tenofovir, abacavir, lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir, zalcitabine, telbivudine and didanosine), protease inhibitors (i.e., darunavir, azanavir, fosamprenavir, tipranavir, ritonavir, nerfumavir, amprenavir, indinavir, and saquinavir), purine nucleosides (i.e., valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir, valganciclovir, and cidofovir), and other antivirals (i.e., enfuvirtide, foscarnet, palivizumab, and fomivirsen), antibiotics (aminopenicillins (i.e., amoxicillin, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, flucoxacillin, temocillin, azlocillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, and bacampicillin), cephalosporins (i.e., cefazolin, cephalexin, cephalothin, cefamandole, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cephradine, loracarbef, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin), carbapenems / penems (i.e., imipenem, meropenem, ertapenem, faropenem, and doripenem), monovaccinia, cephalosporins (i.e., cephazolin, cephalexin, cephalothin, cefamandole, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cephradine, loracarbef, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin), beta-lactams (i.e., aztreonam, tigemonam, norcardicin A, and tabtoxinine beta-lactams), beta-lactamase inhibitors in combination with another beta-lactam antibiotic (clavulanic acid, tazobactam, and sulbactam), aminoglycosides (i.e., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromomycin), ansamycins (i.e., geldanamycin and herbimycin), carbacephems (i.e., loracarbef), glycopeptides (i.e.,teicoplanin and vancomycin), macrolides (i.e., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spectinomycin), monobactams (i.e., aztreonam), quinolones (i.e., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamides (i.e., mafenide, sulfonamide chrysoidim, sulfacetamide, sulfadiazine, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim, and sulfamethoxazole), tetracyclines (i.e., tetracycline, demeclocycline, doxycycline, minocycline, and oxytetracycline), antineoplastic agents (i.e., tetracycline, demeclocycline, doxycycline, minocycline, and oxytetracycline), Antitumor or cytotoxic antibiotics (i.e., doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin), as well as other antibacterial compounds (i.e., bacitracin, colistin, and polymyxin B), antifungal agents (i.e., metronidazole, nitazoxanide, imidazole, chloroquine, iodoquinol, and and paromomycin), and antiparasitic agents (including, but not limited to, quinine, chloroquine, amodiaquine, pyrimethamine, sulfadoxine, proguanil, mefloquine, atovaquone, primaquine, artemesinin, halofantrine, doxycycline, clindamycin, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, rifampin, amphotericin B, melarsoprol, eflornithine, and albendazole). In the case of ischemia, thrombolytic agents (i.e., urokinase, alteplase, reteplase, and tenecteplase), platelet aggregation inhibitors (i.e., aspirin, cilostazol, clopidogrel, prasugrel,and Zipi, Neurologic agents including ritonavir, ritonavir, statins (i.e., lovastatin, pravastatin, foetivastatin, rosuvastatin, atorvastatin, simvastatin, cerivastatin, and pitavastatin), and compounds to improve blood flow or vascular flexibility (including, for example, blood pressure medications) may be selected.

[0064] For behavioral disorders, a neurological agent selected from the following behavior-modifying compounds may be selected, including but not limited to atypical antipsychotics (i.e., risperidone, olanzapine, apripiprazole, quetiapine, paliperidone, asenapine, clozapine, iloperidone, and ziprasidone), phenothiazine antipsychotics (i.e., prochlorperazine, chlorpromazine, fluphenazine, perphenazine, trifluoperazine, thioridazine, and mesoridazine), thioxanthenes (i.e., thiothixene), other antipsychotics (i.e., pimozide, Lithium, molindone, haloperidol, and loxapine), selective serotonin reuptake inhibitors (i.e., citalopram, escitalopram, paroxetine, fluoxetine, and setraline), serotonin-epinephrine reuptake inhibitors (i.e., duloxetine, venlafaxine, desvenlafaxine), tricyclic antidepressants (i.e., doxepin, clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine, imipramine, protriptyline, and desipramine), tetracyclic antidepressants (i.e., mil tazapine and maprotiline), phenylpiperazine antidepressants (i.e., trazodone and nefazodone), monoamine oxidase inhibitors (i.e., isocarboxazid, phenelzine, selegiline, and tranylcypromine), benzodiazepines (i.e., alprazolam, estazolam, flurazeptam, clonazepam, lorazepam, and diazepam), norepinephrine-dopamine reuptake inhibitors (i.e., bupropion), CNS stimulants (i.e., phentermine, diethylpropion, methamphetamine, dextroamphetamine, amphetamine, amphetamine, methylphenidate, dexmethylphenidate, lisdexamfetamine, modafumil, pemoline, phendimetrism, benzphetamine, phendimetrism, armofetrism, diethylpropion, caffeine, atomoxetine, doxapram, and mazindol), anxiolytics / sedatives / hypnotics (barbiturates (i.e., secobarbital, phenobarbital, and mephobarbital), benzodiazepines (listed above), and other anxiolytics / sedatives / hypnotics (i.e., diphenhydramine,Examples of antidepressants include, but are not limited to, sodium oxybate, zaleplon, hydroxyzine, chloral hydrate, aorpidem, buspirone, doxepin, eszopiclone, ramelteon, meprobamate, and ethchlorvynol), secretin (see, e.g., Ratliff-Schaub et al. Autism 9:256-265 (2005)), opioid peptides (see, e.g., Cowen et al, J. Neurochem. 89:273-285 (2004)), neuropeptides (see, e.g., Hethwa et al. Am. J. Physiol. 289:E301-305 (2005)).

[0065] In the case of CNS inflammation, neurological drugs that address the inflammation itself (i.e., nonsteroidal anti-inflammatory drugs such as ibuprofen or naproxen), or drugs that treat the underlying cause of the inflammation (i.e., antiviral or anticancer drugs) may be chosen.

[0066] Also provided herein is a pharmaceutical composition comprising a conjugate of an AAV capsid and an effector entity. The pharmaceutical composition described herein is designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. In certain embodiments, the compositions described herein are administered systemically (e.g., intravenously). In one embodiment, direct delivery to the CNS is desired and may be performed via intrathecal injection. The term "intrathecal administration" refers to delivery targeted to the cerebrospinal fluid (CSF). This may be done by direct injection into the ventricular CSF or lumbar CSF, suboccipital puncture, or other suitable means. Meyer et al., Molecular Therapy (October 31, 2014) demonstrated the efficacy of direct CSF injection, resulting in widespread transgene expression throughout the spinal cord in mice and non-human primates using a 10-fold lower dose compared to IV application. This document is incorporated herein by reference. In one embodiment, the composition is delivered via intraventricular viral injection. See, e.g., Kim et al., J Vis Exp. 2014 Sep 15;(91):51863, incorporated herein by reference. See also, Passini et al., Hum Gene Ther. 2014 Jul;25(7):619-30, incorporated herein by reference. In another embodiment, the composition is delivered via lumbar injection.

[0067] Alternatively, other routes of administration (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, intraperitoneal, and other parenteral routes) may be selected. Thus, the pharmaceutical composition may be formulated (e.g., in the form of a liquid solution or suspension) for any suitable route of administration (e.g., for intravenous administration, for oral administration, etc.). Alternatively, the pharmaceutical composition may be in a solid form (e.g., a tablet or capsule) (e.g., for oral administration). In some embodiments, the pharmaceutical composition may be in the form of a powder, a drop, an aerosol, etc.

[0068] The term "pharmaceutical formulation" refers to a preparation that is in a form that is effective for the biological activity of the active ingredient contained therein and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0069] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0070] Methods and agents well known in the art for making formulations are described, for example, in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Easton, Pa. Formulations may contain, for example, excipients, carriers, stabilizers, or diluents (such as sterile water, saline, polyalkylene glycols (such as polyethylene glycol), oils of vegetable origin, or hydrogenated naphthalenes), preservatives (such as octadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol), alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides, proteins (such as serum albumin, gelatin, or immunoglobulins), and the like. The surfactant may include immunoglobulins, hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, and lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, and dextrins), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants (TWEEN™, PLURONICS™, or polyethylene glycol (PEG)).

[0071] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0072] The conjugate of AAV capsid and effector entity described herein can be used in various in vivo methods.In one embodiment, a method for treating neurological disease is provided, which comprises administering the conjugate described herein.Thus, the binding of AAV capsid to GPI anchor BBB ligand promotes the delivery of the effector entity conjugated thereto to the CNS, and the BBB ligand functions as a receptor or co-receptor that mediates the viral entry or transport of the conjugate across the BBB.

[0073] "Administration" or "route of administration" refers to delivery of a composition described herein to a subject, with or without a pharmaceutical carrier or excipient. Routes of administration may be combined, if desired. In some embodiments, administration is repeated periodically. In certain aspects, the compositions described herein are co-administered. In certain aspects, an AVV vector or conjugate of the invention is co-administered with an antibody. When compositions are co-administered, they may be formulated together or separately and delivered to a subject essentially simultaneously by the same or different routes. In certain aspects, the time between administering a composition to a subject may be less than 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, or 48 hours. In certain embodiments, a composition described herein is administered to a subject in need thereof one or more times. In certain embodiments, the composition may be allowed to be re-administered one, two, three or more times. In certain aspects, in which a composition comprising an AAV capsid is administered, such re-administration may be of the same type of vector, or of a different vector.

[0074] In certain embodiments, the AAV capsid of the conjugate provided herein comprises an expression cassette, so that the delivery of the vector to the CNS results in the expression of heterologous genes.Thus, the delivery of the AAV capsid to the CNS leads to the ectopic or altered expression of gene products, which benefits patients with neurological disorders.

[0075] "Change in expression" or "altered expression" or similar phrases refer to an upregulation of the expression levels of a nucleic acid sequence (e.g., a gene or transcript) or cell surface marker relative to a selected reference standard or control, or a downregulation of the expression levels of a nucleic acid sequence (e.g., a gene or transcript) or cell surface marker relative to a selected reference standard or control, or a combination of a pattern or relative pattern of particular upregulated and / or downregulated genes and / or cell surface markers. The extent of altered expression may differ for each individual gene or marker or may vary between subjects. Thus, in certain embodiments, delivery of a vector or effector entity described herein across the BBB results in altered expression of a CNS target (i.e., gene, protein, etc.).

[0076] In certain embodiments, the methods described herein are useful for treating lysosomal storage diseases, which are metabolic disorders that in some cases have CNS-related or CNS-specific symptoms, including, but not limited to, Tay-Sachs disease, Gaucher disease, Fabry disease, mucopolysaccharidoses (types I, II, III, IV, V, VI, VII), glycogen storage diseases, GM1 gangliosidosis, metachromatic leukodystrophy, Farber disease, Canavan leukodystrophy, and neuronal ceroid lipofuscinosis types 1 and 2, Niemann-Pick disease types A and B (acid sphingomyelinase deficiency or ASMD), Niemann-Pick disease type C (NPC), Pompe disease, and Krabbe disease.

[0077] In another embodiment, a method of altering the infectivity of the CNS by an AAV vector is provided. In one aspect, a combination therapy is provided, in which the AAV capsid is ligated to a GPI anchor BBB ligation. The present invention includes co-administering the antibody or antibody fragment with a binding partner of the antibody and an antibody or antibody fragment that binds to a BBB ligand. Thus, the antibody or antibody fragment binds to a GPI-anchored ligand, thereby reducing or inhibiting viral entry at the BBB. The reduction or inhibition of CNS infectivity can be measured relative to the incidence observed in the absence of the co-administered antibody or antibody fragment. In certain embodiments, infectivity in the presence of the antibody can be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to infectivity in the absence of the antibody.

[0078] Also provided herein is a method for engineering AAV capsids that are particularly useful for targeting the CNS.Engineered capsids include peptide fragments (i.e., binding partners) that specifically bind to GPI-anchored proteins on the BBB.Thus, AAV capsids can be engineered to confer or enhance binding to GPI-anchored proteins selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B. In one embodiment, the method involves identifying a peptide fragment that specifically binds to a GPI-anchored protein and an amino acid sequence encoding such a binding partner, and then modifying the AVV capsid to express the binding partner, thereby generating an AVV capsid that is capable of binding to a GPI-anchored ligand on the BBB or has enhanced binding to the GPI-anchored ligand. In one embodiment, the method comprises identifying a binding partner of a ligand selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B. In another embodiment, the method comprises modifying the AAV HVRVIII site or another HVR site to express a binding partner of a GPI-anchored ligand on the BBB. In yet another embodiment, the AAV is selected from AAV1, AAV3B, or AAV9, and the engineered capsid specifically binds to a GPI-anchored protein on the BBB.The engineered AAV capsids obtained by these methods can be used for conventional gene therapy (i.e., delivery of expression cassettes) and / or can be conjugated to effector entities as described herein.

[0079] In another embodiment, the conjugate described herein is utilized in a method for delivering detectable effector entities to the CNS of a subject. In one aspect, delivery of detectable entities allows for quantifying CNS targets to diagnose subjects with neurological disorders. In one aspect, the detectable agent binds to the CNS target and comprises one or more of a peptide, a nucleic acid, an siRNA, an antibody, an antibody fragment, a small molecule, a lipid nanoparticle, or a cytotoxic agent. Thus, in certain aspects, the method provided herein comprises administering a detectable agent and imaging the CNS, for example, using magnetic resonance imaging (MRI) or computed tomography (CT).

[0080] These examples are provided for illustrative purposes only. The compositions, experimental protocols, and methods disclosed and / or claimed herein can be made and executed without undue experimentation in light of the present disclosure. The protocols and methods described in the examples are not to be construed as limitations on the scope of the invention as claimed. Rather, the present The specification should be construed to encompass any and all modifications that become evident as a result of the teachings provided herein. Those skilled in the art will understand that modifications or variations can be made in the disclosed embodiments of the examples and still obtain similar expected results. For example, the substitution of chemically or physiologically related reagents for the reagents described herein is expected to produce the same or similar results. All such similar substitutions and modifications are apparent to those skilled in the art and are within the scope of the present invention. EXAMPLES

[0081] Example 1: GPI-linked protein LY6A (SCA-1)-mediated transport across the blood-brain barrier The factors that enable AAV-PHP.B to cross the blood-brain barrier (BBB) ​​with such efficiency have not been previously defined. In this study, the high BBB permeability of AAV-PHP.B was specific to C57BL / 6J mice and is consistent with previous work investigating its role in hematopoietic, mesenchymal, and cancer stem cell biology. 9-12 This was determined based on the specific binding of the seven inserted amino acids to a GPI-anchored protein expressed on brain endothelial cells called LY6A (also known as SCA-1), which has been studied in the context of

[0082] method animal All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania, and animals were housed in an AAALAC-accredited barrier facility within the School of Medicine at the University of Pennsylvania. The University of Pennsylvania Office of Laboratory Animal Welfare (OLAW) assurance number is A3079-01. C56BL / 6J (strain #000664), BALB / cJ (#000651), and F1 hybrid CB6F1 / J (#100007) were purchased from Jackson Laboratory. F2 hybrids were obtained by crossing CB6F1 / J in our facility. Ly6a-null mice on C57BL / 6J and BALB / cJ backgrounds were generously provided by William L. Stanford (University of Ottawa). For reporter gene experiments, adult (6–8 weeks old) males were injected. Animals were housed in standard cages, 2–5 per cage. Cages, water bottles, and bedding in the barrier facility were autoclaved and cages were changed once a week. An automatically controlled 12-h light-dark cycle was maintained. Each dark period began at 19:00 h (± 30 min). Irradiated laboratory rodent chow was provided ad libitum.

[0083] cell line Human Embryonic Kidney 293 cells (HEK293, originally obtained from female fetuses) were maintained in Dulbecco's Modified Eagle's Medium (DMEM Gibco, ThermoFisher Scientific, cat# 11995-040) supplemented with 10% gamma-irradiated fetal bovine serum (Hyclone™, cat#SH30071.03IR) and 100 IU / mL penicillin / streptomycin and grown at 37°C in a humidified incubator containing 5% CO2.

[0084] Vector production The AAV9.PHP.B transformant plasmid (pAAV2 / PHP.B) was generated using pAAV2 / 9 (Penn Vector Core) as a template with the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Cat. No. 210515) according to the manufacturer's manual. AAV-PHP.B mutants were also constructed in the same manner using pAAV2 / PHP.B as a template, as previously described. 37 AAV vectors were produced and titrated by the Penn Vector Core. Briefly, HEK293 cells were transfected in triplicate, and culture supernatants were harvested, concentrated, and purified using an iodixanol gradient. The purified vector was purified using the plasmid p53A-expressing vector ... 38 , and titrated by digital droplet PCR using primers targeting the rabbit β-globin polyA sequence.

[0085] In vivo studies and histology Mice were administered 1 × 10 vectors encoding enhanced GFP (Penn Vector Core) in 0.1 mL via the lateral tail vein. 12 GC(5×10 13Mice received 100 mg / kg of AAV9 or AAV-PHP.B or AAV-PHP.eB vectors and were euthanized by CO2 inhalation 21 days after injection. Brains and other tissues were rapidly harvested. Sagittally cut half of the brain was immersion fixed in 10% neutral buffered formalin for approximately 24 hours, briefly washed in PBS, and equilibrated sequentially in 15% and 30% sucrose in PBS at 4°C. Tissues were then frozen in OCT embedding medium and cryosectioned for direct GFP visualization (brains were sectioned at 30 μm thickness, other tissues were sectioned at 10 μm thickness). Images were taken using a Nikon Whole brain sections were acquired with an Eclipse Ti-E fluorescence microscope or scanned with an Aperio Versa slide scanner. The other half of the brain was either snap frozen on dry ice for qPCR vector biodistribution studies or formalin-fixed and paraffin-embedded for immunostaining. Immunofluorescence for LY6A was performed on formalin-fixed paraffin-embedded brain samples. Sections were deparaffinized and boiled for 6 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval, then blocked for 15 min with 1% donkey serum in PBS + 0.2% Triton, followed by sequential incubation with primary antibodies (1 h) and fluorescently labeled secondary antibodies (45 min) diluted in blocking buffer. Monoclonal rat antibody D7 against LY6A (eBioscience, ThermoFisher Scientific Cat#14-5981-82) was used at a dilution of 1:200, and TRITC-labeled donkey anti-rat (Jackson Immunoresearch Cat#712-025-153, dilution of 1:100) served as the secondary antibody.

[0086] Vector biodistribution Tissue DNA was extracted with a QIAamp DNA Mini kit (Qiagen catalog no. 51306), and vector genomes were quantified by real-time PCR using Taqman reagents (Applied Biosystems, Life Technologies) and primers / probes targeting the rBG polyadenylation sequence of the vector.

[0087] Whole-exome sequencing Whole-exome sequencing libraries were generated from genomic DNA isolated from the brains of 4 F1 and 12 F2 mice using the Agilent SureSelect Mouse All Exon Kit (Agilent catalog number 5190-4641). Samples were indexed and sequenced on a NextSeq high-output cartridge (8 samples per flow cell). After sequencing, paired-end reads from each sample were analyzed for whole-exome sequencing using NovoAlign (v3.08.02). 39 The sequences were mapped to the reference genome (GRCm38) using parameters optimized for . Duplicate reads (optical and / or PCR duplicates derived from a single fragment of DNA) were then flagged with Picard tools (v2.13.2). After data preprocessing, the GATK best practices were used to identify the sequences. 40~43 Variant calling was performed for each sample using GATK (v3.8). Briefly, base quality score recalibration was first performed, followed by variant calling and joint genotyping using GATK (v3.8). Raw variants were then filtered to remove Mendelian violation variants and variants with a QUAL score below 50. High confidence variants were then used for association testing (see Statistical Analysis). Snp Effect 44 was used to annotate genomic variants and predict functional effects.

[0088] Construction of Ly6a expression vector LY6A from C57BL / 6J and BALB / cJ coding sequences were synthesized as GeneArt strings (ThermoFisher Scientific). For native LY6A expression vectors, GeneArt strings were assembled into BamHI-digested pcDNA3.1(+)IRES GFP (Addgene 51406) using NEBuilder HiFi DNA Assembly Master Mix (NEB Catalog No. E2621). For Twin-Strep-tagged LY6A expression vectors, DNA encoding the first 111 amino acid residues of LY6A was PCR amplified and then assembled into Esp3I-digested pESG-IBA103 using NEBuilder HiFi DNA Assembly Master Mix (NEB Catalog No. E2621). All constructs were verified by Sanger sequencing.

[0089] Production and purification of Twin-Strep-tagged LY6A HEK293 cells were transiently transfected with a plasmid expressing Twin-Strep tagged LY6A (C57BL / 6J or BALB / cJ variants) using a DNA:polyethyleneimine (PEI-linear polyethyleneimine hydrochloride (molecular weight 40,000), Polysciences, Cat. No. 24765-1) w / w ratio of 1:2. 72 hours after transfection, cell culture supernatants were filtered through a 0.22 μm filter and adjusted to pH 8.0 by adding 1 / 10 volume of 10× buffer W (1 M Tris-HCl (pH 8.0), 1.5 M NaCl, 10 mM EDTA). Transfected 293 cells were lysed in 1× buffer W supplemented with 0.1% TritonX-100 and passed twice through a 27-gauge needle. Biotin depletion was performed by combining cell lysates and culture supernatants, followed by incubation with 1 / 400 volume of BioLock biotin blocking solution (IBA Life Sciences, Cat. No. 2-0205-050) for 15 min, followed by centrifugation. Purification of Twin-Strep tagged LY6A protein was achieved by Strep-Tactin XT affinity chromatography according to the manufacturer's protocol (IBA Life Sciences). Briefly, Strep-Tactin XT Superflow resin (Cat. No. 2-4010-010) was incubated with cell lysates and supernatants for 2 h at room temperature, washed with 4 column volumes (CV) of 1x buffer W, and eluted with 0.6 CV, 1.6 CV, and 0.8 CV of 1x buffer BXT. Elution fractions containing recombinant LY6A were pooled and dialyzed four times in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl. Protein concentration was determined by BCA assay (Pierce, catalogue no. 23225).

[0090] Enzyme-linked immunosorbent assay (ELISA) Strep-TactinXT-coated microplates (IBA Life Sciences, Cat. No. 2-4101-001) were incubated overnight at 4°C with 200 μL of coating buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) containing 0 μg or 0.5 μg of Twin-Strep-tagged LY6A protein per well. Plates were washed three times with PBST (0.05% Tween-20 in PBS) and blocked for 2 h at room temperature in 3% BSA in PBS. AAV serotypes were diluted to the indicated concentrations in PBS supplemented with 1% BSA and 0.1% Pluronic F-68. 200 μL of AAV dilutions were added to each well and incubated at 37°C for 2 h. Immobilized AAV particles were incubated with rabbit antiserum against AAV9 (1:50,000, Penn Immunology Core) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5,000 LY6A-dependent AAV binding was detected by sequential incubation with LY6A-coated wells (Cat. No. 31460, ThermoFisher Scientific) for 1 hour. Plates were developed using 200 μL of SureBlue TMB one-component microwell peroxidase substrate (Seracare, Cat. No. 52-00-01) according to the product instructions, and optical density (OD) was measured at 450 nm by a microplate reader (SpectraMax M3). To calculate LY6A-dependent AAV binding, background AAV binding to uncoated microplate wells was subtracted from AAV binding to LY6A-coated wells observed at each virus concentration. Data are representative of three independent experiments. To estimate the apparent affinity (Kd) of the AAV-PHP.B variants for the LY6A variants, a least-squares fit of the ELISA data (taking the A450 signal as a percentage of maximum) was applied to a simple 1:1 model of molecular interactions [f=At / (Kd+At)], where f is the percentage of Ly6a bound and At is the total amount of AAV applied to the well. A more complex model explicitly accounting for Ly6a did not improve the fit. The ELISA orientation used only allows us to estimate the apparent affinity of the vector for the LY6A protein as seen in cells, and does not represent the microscopic affinity of a single PHP.B peptide loop with a single LY6A protein.

[0091] HEK293 transduction assay All cell cultures were performed in a humidified incubator at 37°C, 5% CO2 using DMEM medium + 10% heat-inactivated fetal bovine serum (FBS) + 1% penicillin-streptomycin (P / S). On the first day of the assay, HEK293 cells were trypsinized, counted, and seeded in 96-well plates (Corning #3603) at a density of 80,000 cells / well. After 20-24 hours, cells were transiently transfected with plasmid constructs containing C57BL / 6J or Balb / cJ alleles of Ly6a or Ly6c1 upstream of the IRES2-EGFP sequence. Transfections were performed in serum-free DMEM using 0.14ug of plasmid DNA and 0.28ug of PEI per well in a culture volume of 100uL. Mock-transfected cells received only PEI in serum-free DMEM. 24 hours after transfection, each well was supplemented with 100uL of DMEM+20% FBS+1% P / S and grown for another 24 hours under full serum conditions. 48 hours after transfection, GFP expression was qualitatively assessed in the transfected wells using a fluorescent microscope. AAV9 and AAV9-PHP.B viral vectors containing a β-galactosidase reporter gene under the control of the CMV promoter were then introduced into each well at MOIs ranging from 100,000 to 10. This transduction step was performed for 2 hours in 100uL of serum-free DMEM, followed by the addition of 100uL of DMEM+20% FBS+1% P / S and incubation for 24 hours. β-galactosidase expression was then determined using the Galacto-StarB-Galactosidase Reporter Gene Assay System (Thermo-Fisher Scientific, #T1014) following the manufacturer's direct lysis protocol for microplate cultures. Lysis was performed for 30 minutes using 40uL of lysis buffer per well with some modifications to the lysis step of this protocol. Luminescence detection was then performed on a SpectramaxM3 luminescence plate reader. Data are representative of six independent experiments.

[0092] HEK293 antibody inhibition assay The HEK293 antibody inhibition assay was performed as described above for the HEK293 transduction assay with the following modifications or additions: Only C57BL / 6J and Balb / cJ Ly6a-IRES2-EGFP plasmids were used for transfection. In addition, an antibody incubation step was added on day 4 of the assay, prior to transduction with AAV9-PHP.B. This step was performed using low D7 endotoxin-free azide-free 100% ethanol. Anti-LY6A antibody (Abeomics, #31-2027) or IgG isotype control (Abcam, #18450) was incubated with cells for 1 hour at 4° C. Antibodies were introduced at 100 nM in 50 uL of serum-free DMEM, and after this incubation, AAV9-PHP.B reporter vector was introduced at an MOI of 10,000 in 50 uL of serum-free DMEM. Plates were then returned to a 37° C. incubator and the remainder of the assay proceeded as previously described. Data are representative of 8 independent experiments.

[0093] Quantitative and statistical analysis Vector genome copies in mice were analyzed using one-way ANOVA (Kruskal-Wallis test) followed by Dunn's multiple comparison test (GraphPad Prism) with an alpha value of 0.05. For WES linkage analysis, trait-associated variants were identified using a linear Wald test for quantitative traits (https: / / github.com / statgen / EPACTS). Only variants with p-values ​​≤ 5E~8 were considered significant. HEK293 transduction efficiency (β-galactosidase activity) was compared using two-way ANOVA followed by mean comparison test with Tukey's multiple comparison test (GraphPad Prism).

[0094] result BBB permeability to AAV-PHP.B is inherited as a codominant trait in mice. AAV-PHP.B carrying the GFP transgene was added at 1 × 10 12 Genome copy (GC) IV administration to BALB / cJ mice 13It was previously shown that AAV-PHP.B resulted in widespread transduction of cells of the central nervous system (CNS) in C57BL / 6J mice but not in BALB / cJ mice. In contrast, direct administration of AAV-PHP.B to the CNS by intracerebroventricular injection, bypassing the BBB, resulted in comparable, robust GFP expression in both C57BL / 6J and BALB / cJ mouse brains (Figure 5A and Figure 5B). Thus, we conclude that although cells of the CNS of both strains were susceptible to AAV-PHP.B intracerebral transduction, the increased efficiency of AAV-PHP.B in C57BL / 6J mice was due to enhanced delivery across the BBB. We hypothesized that the strain-specific differences in AAV-PHP.B permeability of the BBB were caused by genetic variations in a single gene involved in BBB transport. To confirm this hypothesis, we assessed CNS transduction in F1 and F2 offspring of a C57BL / 6JxBALB / cJ mating by IV administration of AAV-PHP.B. All F1 progeny showed moderate CNS transduction compared to the parental strains, whereas the F2 generation showed a distribution of transduction, with 55.5% having moderate, 16.7% having high and C57BL / 6J-like, and 27.8% having low and BALB / cJ-like CNS transduction (Figure 1A). This result was confirmed by qPCR-based quantification of the genomic copy number of the vector in the corresponding mouse brains (Figure 1B). Based on the distribution of F1 and F2 phenotypes, we concluded that the permeability of the BBB to PHP.B follows a Mendelian inheritance pattern of two codominant alleles at a single genomic locus. In contrast to AAV-PHP.B, no strain-specific intracerebral transduction was observed in the case of AAV9 (Figure 1B).

[0095] The LY6A gene is associated with transduction of PHP.B across the BBB. The inheritance pattern of CNS transduction described in the F1 and F2 offspring suggests that variation in a single gene may underlie the strain-specific differences in this phenotype (i.e., the high BBB penetration of AAV-PHP.B). Therefore, to identify any causative mutations within the coding region of the mouse genome, we performed whole exome sequencing (WES) based on genotyping and genetic linkage analysis of 16 related mouse strains (Figure 2A). Our analysis revealed that approximately 1000 karyotype mutations spanning the D3 and E3 karyotype bands of mouse chromosome 15 were most significantly associated (p=1.9E-31) with the observed phenotype (Figure 2B). We identified 135 unique mutations located within a 4.5 Mbp genomic DNA stretch. Functional variant annotation of the significant variants identified revealed that missense mutations within the Ly6a, Ly6i, Rhophilin1, and Riken cDNA2010109I03 genes were most significantly linked to high BBB permeability of AAV-PHP.B (Table 1). Subcellular localization of the proteins encoded by these genes and public databases 14 Based on their abundance in the brain by , we hypothesized that LY6A (also known as SCA-1), a GPI-anchored surface protein highly expressed in the brain microvasculature, was the protein responsible. To test whether Ly6a was essential for the highly efficient delivery of AAV-PHP.B across the BBB, AAV-PHP.B carrying a GFP transgene was transfected into mice with a Ly6a knockout (Ly6a) in a C57BL / 6J background and wild-type controls. - / - ) mice were injected IV. - / - We observed that the liver was efficiently transduced, but the brain was minimally transduced in mice (Figure 2C), indicating that LY6A is required for AAV-PHP.B transport across the BBB. Interestingly, a polymorphism in the Ly6 gene cluster reduced the expression of Ly6 a (BALB / cJ-like) and Ly6 bIt has been previously described that the BRCA1 gene has two major haplotypes, one with a BRCA1 gene and the other with a BRCA1 gene (C57BL / 6J-like). These two haplotypes differ with respect to several single nucleotide changes in the promoter region and two amino acid substitutions in the LY6A protein (i.e., Ly6 a The protein encoded by Ly6 b Analysis of brain tissue by immunohistochemistry with an antibody against LY6A revealed that its expression was at high levels in endothelial cells of microvessels of C57BL / 6J animals and was greatly reduced in BALB / cJ animals (Figure 2D), which is consistent with previous reports. 15 This is to confirm that - / - In tissues from mice, no expression of LY6A was detected, confirming the specificity of the assay (Figure 2D). Either a mutation in the promoter or a mutation within the open reading frame of LY6A in BALB / cJ may contribute to the dramatic reduction in LY6A expression on brain endothelium.

[0096] Based on these observations, the strain-specific BBB permeability of AAV-PHP.B observed in C57BL / 6J and BALB / cJ mice may be due to the involvement of Ly6 b and Ly6 a We hypothesized that the results of this study could be generalized to all strains of mice that carry the Ly6 haplotype. To support this hypothesis, six additional inbred strains of mice were tested, three of which contained the Ly6 b haplotypes (129S1 / Svlmj, DBA2 / J, and FVB / NJ), and three pedigrees were a The mice had three haplotypes (C3H / HeJ, CBA / J, and A / J) (Figure 3A). Mice from each strain were injected IV with AAV-PHP.B expressing GFP and analyzed for CNS transduction. As expected, high-level CNS transduction was observed in mice expressing Ly6 b(C57BL / 6J-like) haplotype (Figure 3B). Taken together, we conclude that variants in the Ly6a gene are associated with PHP.B transduction across the BBB. [Table 1-1] [Table 1-2]

[0097] AAV-PHP.B and AAV-PHP.eB bind to the LY6A protein with high affinity To evaluate the possibility of a direct interaction between LY6A variants and the AAV capsid, an ELISA assay was developed. For both C57BL / 6J and BALB / cJ variants, expression cassettes containing a GPI-anchor truncated version of LY6A were constructed to allow the isolation of a soluble version of the respective recombinant protein. The ELISA assay was developed by analyzing the binding of AAV particles to recombinant LY6A protein bound to ELISA wells (Figure 4A). More AAV.PHP.B bound to C57BL / 6J LY6A than to BALB / cJ LY6A. No binding between AAV9 and LY6A variants was detected (Figure 4A). Despite the lower signal intensity of AAV-PHP.B to BABL / cJ LY6A, a relatively high affinity binding remained. Indeed, both data sets fit well to sub-nM binding isotherms (0.07 nM and 0.28 nM for C57BL / 6J and BALB / cJ variants, respectively). This affinity was suspected to represent a strong association between multiple immobilized Ly6a proteins and the AAV-PHP.B vector. The reduced BBB transport of AAV-PHP.B in BABL / cJ may be caused by reduced binding to LY6A and reduced expression of LY6A.

[0098] Valine 592 of the AAV9-PHP.B capsid, located within the 588-TLAVPFK peptide insert, was subjected to saturation mutagenesis. Each vector variant was purified and individually tested for binding affinity to LY6A using SPR. Biacore sensograms identified high-affinity, native-affinity, and low-affinity variants of AAV9-PHP.B (Figure 8A-C).

[0099] A variant with a single amino acid mutation in the seven amino acid loop of AAV-PHP.B We generated a mutant capsid, AAV-PHP.B V592G (a valine to glycine mutation at position 592 of VP1 in the fourth residue of the seven inserted amino acids). The expression profile following IV injection of a GFP-expressing version of this mutant capsid is similar to AAV9 but not to AAV-PHP.B (e.g., high transduction in the liver versus little transduction in the CNS) (Figure 6). Interestingly, neither AAV9 nor AAV-PHP.B V592G detectably bound LY6A (Figure 4A), providing a direct link between capsid interaction with LY6A in vitro and the ability to cross the BBB in vivo.

[0100] We then examined the ability of LY6A to enhance AAV capsid internalization. In this assay, the full-length Ly6a gene was transiently transfected into HEK293 cells and then incubated with low- and high-affinity capsids, including AAV-PHP.B or AAV9 expressing lacZ. In this assay, transduction of lacZ serves as a surrogate for internalization. Transduction, as measured by β-galactosidase activity in cell lysates, was achieved in a dose-dependent manner for AAV-PHP.B incubated with cells expressing C57BL / 6J or BALB / cJ LY6A proteins (Figure 4B). No transduction above background was achieved in the case of AAV9 in cells expressing any version of LY6A or in the case of AAV-PHP.B in cells expressing the unrelated GPI-anchored protein LY6C1 (Figure 4B). The low affinity variant shows a modest increase (2-fold) in transduction efficiency compared to the high affinity variant at low MOI. Binding to the Ly6a receptor at both levels is sufficient to improve transduction efficiency by more than 10-fold compared to AAV9 (Figure 9). Furthermore, enhanced PHP.B transduction of C57BL / 6J or BALB / cJ cells expressing the LY6A protein was blocked by preincubating the cells with an anti-LY6A rat monoclonal antibody (but not by preincubation with an isotype control), further strengthening the hypothesis of a role for a co-receptor or receptor for LY6A (Figure 4C).

[0101] A second generation version of AAV-PHP.B, called AAV-PHP.eB, was recently described and showed even higher CNS transduction in C57BL / 6J mice after IV injection. This variant was investigated to determine whether LY6A is a major determinant of BBB permeability. After IV injection of AAV-PHP.eB, CNS transduction was observed in C57BL / 6J mice, but not in BALB / cJ mice as observed with AAV-PHP.B (Figure 7).

[0102] AAV-PHP.eB bound to LY6A in C57BL / 6J with a signal slightly higher than that achieved with AAV-PHP.B (Figure 4A). It was concluded that interaction with LY6A remains important for the neurotropic properties of both AAV-PHP.B and AAV-PHP.eB.

[0103] Effect of LY6A binding affinity on biodistribution AAV9-PHP.B affinity variants were prepared with the CB7-eGFP reporter gene and tested in C57BL / 6 mice at an IV dose of 1e12gc / mouse. Mice were euthanized 21 days after injection and organs were harvested for histology and biodistribution. Ly6a affinity correlates negatively with liver biodistribution, with vectors with the strongest affinity for Ly6a showing reduced liver localization. This trend is not observed in brain biodistribution. All vectors with affinity for the LY6A receptor show comparable levels of brain tissue localization (Figure 10A). When biodistribution was followed by liver expression of eGFP, variants with low affinity for Ly6a show increased liver tissue localization and expression. Brain histology showed that high and low affinity vectors have similar biodistribution in brain tissue, whereas vectors with low to moderate affinity for LY6A show increased liver tissue localization and expression. The vectors that were used showed improved expression (FIG. 10B).

[0104] Valency of PHP.B peptide presentation and effects on cell transduction Chimeric capsids were produced by altering the ratio of plasmids encoding either the AAV9 capsid gene or the AAV9-PHP.B capsid gene used during vector production. The transduction efficiency of each chimeric variant was quantified by expression of β-galactosidase in HEK293 cells stably expressing Ly6a. Capsids displaying PHP.B peptides at a 1:3 PHP.B, 2:3 AAV9 ratio transduced cells comparably when compared to capsids displaying 100% PHP.B peptide. Also, modulating the valency of PHP.B peptide display resulted in graded responses in transduction, whereas modulating peptide affinity (Figure 11) resulted in more binary responses in transduction.

[0105] Binding between soluble LY6A and PHP.B requires multivalent display of LY6A domains. Previous binding and affinity SPR data was generated with LY6A immobilized on the sensor chip surface and AAV9-PHP.B in solution on the sensor surface. In this orientation, the vector associates with multiple surface receptors and avidity effects enhance the binding interaction. In the reverse orientation, monomeric LY6A is unable to bind to surface immobilized AAV9-PHP.B even at concentrations up to 40 μM. When LY6A is expressed as a dimer fused to the N-terminus of an IgG1-Fc domain, it is still unable to bind to surface immobilized AAV9-PHP.B at a concentration of 1 μM. When LY6A is expressed as an N-terminal fusion to an IgM-Fc domain, up to 12 copies of the Ly6a domain can be presented on a single molecule. With this construct, specific binding to AAV9-PHP.B, but not AAV9, can be observed (Figure 12).

[0106] Consideration As gene transfer technologies have advanced, so has our understanding of the key drivers of performance, such as transduction efficiency and host-vector response. Some of these translational studies have enhanced our understanding of the fundamental biological processes involved. The ability of AAV vectors to transduce cells of all mammalian CNS in a targeted and dose-dependent manner has revolutionized neurobiology research through the use of tools such as optogenetics ( 16 (Reviewed in ). Studies designed to blunt the adaptive immune response to adenovirus-based in vivo gene transfer have uncovered a mechanism by which T cells are activated through the association of CD40 with CD40L. 17 The story described in this paper is another example of translational work illuminating new biological principles with broader implications than those related to gene therapy.

[0107] The isolation of the AAV9 variant AAV.PHP.B heralds a potentially transformative approach to the treatment of neurological diseases: the efficiency with which CNS cells can be transduced after IV injection in C57BL / 6J mice is at least 20-fold higher than previously achieved, which, when transposed to primates, will enable the development of therapeutics across a broad range of neurological diseases. 18 Initial evaluation of AAV-PHP.B demonstrated that its BBB permeability was similar to that of C57BL / 6J mice. 13 This led to the unexpected discovery that the BBB permeability is restricted to a few strains of mice, such as C57BL / 6J and BALB / cJ. The substantial differences in the biology of transduction between two related but genetically distinct strains of mice, C57BL / 6J and BALB / cJ, allowed us to use a classical genetic linkage approach to determine that just one gene determines this dramatic strain difference in BBB permeability, and that it encodes the GPI-anchored protein, LY6A.

[0108] LY6A (also called Sca-1) is an antigen that is upregulated on activated lymphocytes. It was discovered over 40 years ago as a marker for hematopoietic stem cells (HSCs) in adults and is commonly used to enrich for adult mouse hematopoietic stem cells (HSCs) and is also expressed in stem, progenitor, and differentiated cell types in a wide variety of tissues and organs ( 9 (Reviewed in ). It is intriguing that, despite its common use in stem cell biology research, no ligand for LY6A has been identified. 19~21 Its physiological function is also unknown, but studies in Ly6a null mice have demonstrated that it downregulates T cell proliferation. 22 , Hematopoietic lineage regulation, HSC engraftment and homing 10 , mesenchymal stem cell self-renewal, and osteogenesis 23,24 Here, we identify for the first time a ligand for LY6A (i.e., the AAV9 capsid variant AAV.PHP.B) and demonstrate a direct link between the binding of this ligand to LY6A and its ability to cross the BBB. LY6A mediates the regulation of cerebral microvasculature. 14,15,25 It is highly expressed in b The haplotype was previously shown to be associated with lethal wild-type murine adenovirus-1-induced encephalitis. 26,27 In this context, our results suggest that this mouse GPI-anchored protein may play a role in viral interactions and transcytoplasmic transport at the BBB.

[0109] Studies of other GPI-anchored proteins suggest a mechanism by which LY6A may enhance BBB permeability of AAV vectors. GPI-anchored proteins are often localized to lipid rafts, which are dynamic microdomains within the plasma membrane that are enriched with cholesterol, sphingolipids, and a specific set of important signaling molecules, such as receptors and protein tyrosine kinases. Apical-to-basolateral delivery of raft-associated GPI-anchored proteins may facilitate the transport of AAV vectors in polarized cells. 28 Furthermore, transcytoplasmic transport of macromolecules at the BBB occurs, in part, through tightly regulated caveolae-associated lipid rafts. 29It is known that viral entry and exit occurs via the endothelial cell membrane. Several GPI-anchored proteins and lipid rafts in general play a role in the entry and exit of viral particles into cells ( 30 Interestingly, group B coxsackieviruses exhibit a cellular endocytosis by 1) interacting with the apically localized GPI-anchored protein CD55 / decay-accelerating factor (DAF), 2) activating CD55 clustering and Ab1 kinase-driven Rac-dependent actin reorganization, and 3) translocation to lateral tight junctions where the virus particles associate with the receptor CAR and undergo endocytosis. 31 AAV-PHP.B transduction across the BBB involves a three-step mechanism involving the initiation of capture of viral particles and the induction of viral transport to receptors embedded in tight junction regions. In this model, GPI-anchored proteins allow the initial capture of viral particles and trigger viral transport to receptors embedded in tight junction regions. In the case of AAV-PHP.B transduction across the BBB, further experiments are required to determine whether LY6A is a coreceptor that promotes colocalization of viral capsids with other factors or whether it functions as a primary receptor via cross-linking-activated endocytosis.

[0110] Experimental data suggest that AAV9 can cross the BBB without compromising its integrity via transendothelial transport to the basal compartment. 32 However, the efficiency with which this occurs is at least 20-fold lower than that achieved with AAV-PHP.B in C57BL / mice. 8 Other AAV serotypes, such as AAV5, can cross epithelial and endothelial barriers in vitro via transcytoplasmic transport, a phenomenon that is mediated by the administration of tannic acid or filipin, two chemicals that interfere with the transport of GPI-anchored proteins. 33 Interestingly, it has been suggested that recombinant AAV2 uses the clathrin-independent transporter / GPI-anchored protein-enriched endosomal compartment (CLIC / GEEC) endocytic pathway as the major transduction pathway. 34These studies, together with those reported herein, were performed by independent groups and support a central role for GPI-enriched lipid rafts in AAV transcytoplasmic transport and / or transduction. Our results show for the first time that a GPI-anchored protein can function as a co-receptor or receptor for AAV vectors.

[0111] Example 2: Engineering that binds to GPI-anchored proteins to mediate transduction across the BBB Capsid To improve the delivery of biotherapeutics, GPI-anchored proteins expressed on BBB endothelial cells can be hijacked. However, several groups have failed to demonstrate increased CNS transduction following IV injection of AAV-PHP.B in non-human primates. 13,35 , which is likely explained by the absence of an LY6A homolog in primates. 36 Indeed, the only animal model showing enhanced BBB permeability of AAV.PHP.B is one with a similar genetic background as the model from which it was selected (i.e., C57BL / 6J mice), illustrating how the method of selecting novel capsid variants can limit the utility of candidate capsids. Thus, it will also be productive in developing human gene therapy vectors and other protein therapeutics to evaluate the population of variants that bind to GPI-anchored proteins expressed on endothelial cells derived from primates.

[0112] LY6A has no human orthologue, but other LY6 family members and other GPI-anchored proteins are highly expressed on human brain endothelium (Table 2, source: ISH / IHC Human Protein Atlas www.proteinatlas.org) or have increased brain endothelial expression based on mouse single-cell RNAseq data. 3(Table 3). In addition, some human LY6 proteins, such as LY6E, are associated with neurotropic / endothelial flavivirus infections. Therefore, AAV vectors and capsids can be engineered to interact with GPI-anchored proteins expressed on human brain endothelium to mediate efficient transduction and delivery across the BBB. [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2] References 1. Gao, G. et al. Clades of Adeno-associated viruses are widely disseminated in human tissues.J Virol 78,6381-6388,doi:10.1128 / JVI.78.12.6381-6388.2004(2004). 2.Bell,CLet al.The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice.J Clin Invest 121,2427-2435,doi:10.1172 / JCI57367(2011). 3. 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25.Ma,X.,Robin,C.,Ottersbach,K.& Dzierzak,E.The Ly-6A(Sca-1) GFP Transgene is Expressed in all Adult Mouse Hematopoietic Stem Cells.Stem Cells 20,514-521(2002).26.Spindler,K.R.et al.The major locus for mouse adenovirus susceptibility maps to genes of the hematopoietic cell surface-expressed LY6 family.J Immunol 184,3055-3062,doi:10.4049 / jimmunol.0903363(2010). 27.Stier,M.T.& Spindler,K.R.Polymorphisms in Ly6 genes in Msq1 encoding susceptibility to mouse adenovirus type 1.Mamm Genome 23,250-258,doi:10.1007 / s00335-011-9368-9(2012). 28.Polishchuk,R.,Di Pentima,A.& Lippincott-Schwartz,J.Delivery of raft-associated,GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway.Nat Cell Biol 6,297-307,doi:10.1038 / ncb1109(2004). 29.Andreone,B.J.et al.Blood-Brain Barrier Permeability Is Regulated by Lipid Tra nsport-Dependent Suppression of Caveolae-Mediated Transcytosis.Neuron 94,581-594 e585,doi:10.1016 / j.neuron.2017.03.043(2017). 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[0113] (Sequence Listing Free Text) The following information is identified by <223> Below we provide an array containing free text. [Table 4]

[0114] All publications cited herein, as well as U.S. Provisional Patent Application Nos. 62 / 769,652, filed November 20, 2018, and 62 / 914,035, filed October 11, 2019, respectively, are hereby incorporated by reference. Applicants hereby incorporate by reference the Sequence Listing filed herewith. This file is labeled "18-8634PCT_ST25.txt". Although the invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the appended claims.

Claims

1. A composition comprising a recombinant AAV having a capsid that includes a binding partner for a GPI-anchored blood-brain barrier (BBB) ​​ligand and is conjugated to an effector entity.

2. The composition of claim 1 , wherein the ligand is Ly6E.

3. 3. The composition of claim 1 or 2, wherein the ligand is selected from GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

4. The composition of any one of claims 1 to 3, wherein the capsid is an empty capsid.

5. The composition of any one of claims 1 to 4, wherein the capsid further comprises an AAV vector genome encoding a heterologous gene.

6. The composition of any one of claims 1 to 5, wherein the effector entity is a peptide, a nucleic acid, an siRNA, an antibody, an antibody fragment, a small molecule, a lipid nanoparticle, or a cytotoxic agent.

7. The composition of any one of claims 1 to 6, wherein the AAV capsid and effector entity are conjugated via a linker.

8. 1. A method for treating a neurological disease or disorder in a subject in need of such treatment, comprising contacting the BBB of the subject with an AAV having a capsid that includes a binding partner for a GPI-anchored BBB ligand and is conjugated to an effector entity, wherein binding of the capsid to the GPI-anchored BBB ligand mediates transport of the effector entity across the BBB.

9. 9. The method of claim 8, wherein the ligand is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

10. 9. The method of claim 8, wherein the neurological disease or disorder is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, lysosomal storage disease, and traumatic brain injury.

11. The method of claim 8, wherein the effector entity is a peptide, a nucleic acid, an siRNA, an antibody, an antibody fragment, a small molecule, or a cytotoxic agent.

12. The AAV capsid is conjugated to the effector entity via a linker. The method according to claim 8 .

13. A combination therapy for reducing or inhibiting central nervous system (CNS) uptake of a gene therapy vector having an AAV capsid that contains a binding partner for a GPI-anchored BBB ligand, comprising co-administering an antibody or antibody fragment that binds to the BBB ligand with the gene therapy vector.

14. 14. The method of claim 13, wherein the ligand is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

15. 1. A method for engineering an AAV capsid to target the CNS, comprising: a) identifying an amino acid sequence encoding a peptide fragment that specifically binds to a GPI-anchored BBB ligand; b) modifying the AAV HVRVIII site to express said amino acid sequence; The method, wherein the engineered capsid binds to a GPI-anchored BBB ligand.

16. 16. The method of claim 15, wherein the ligand is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

17. The method of claim 15, wherein the modified AAV is AAV1, AAV3B, or AAV9.

18. 16. An engineered AAV capsid that binds to a GPI-anchored BBB ligand, obtained by the method of claim 15.

19. 1. A method for detectably labeling a CNS target, comprising administering an AAV capsid that binds to a GPI anchor ligand on the BBB and is conjugated to a detectable effector entity, wherein the AAV capsid, upon binding to the GPI anchor BBB ligand, transports the detectable effector entity conjugated to it across the BBB.

20. 20. The method of claim 19, wherein the ligand is selected from Ly6E, GRA3, ALPL, BST2, EFNA5, NT5E, DPEP2, GPC1, LYPD5, GPC6, CD14, CA4, GPC5, CD59, TFPI, EFNA1, EFNA3, HYAL2, MELTF, ULBP2, EFNA4, CNTN5, BCAN, RECK, CFC1, SEMA7A, PRNP, LY6E, PRND, PLAUR, CD24A, MMP25, ART3, LYPD1, PIBF1, CAPRIN1, GFRA3, GPIHBP1, MACF1, and SEC24B.

21. 20. The method of claim 19, wherein the detectable effector entity comprises a peptide, a nucleic acid, an siRNA, an antibody, an antibody fragment, a small molecule, a lipid nanoparticle, or a cytotoxic agent.

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